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OBI-1 is a recombinant B-domain deleted porcine factor VIII (FVIII). FVIII treatment in those with haemophilia A may be complicated by the development of anti-FVIII antibodies (inhibitors) leading to a failure to respond to treatment with human FVIII. To compare the pharmacokinetics and safety of a single dose of OBI-1 with Hyate:C in subjects with haemophilia A and inhibitors, subjects were randomized to receive either Hyate:C followed by placebo or placebo followed by OBI-1 in a double-blind fashion. FVIII levels were assayed using both a one-stage coagulation assay (OSCA) and chromogenic assay. Pharmacokinetic parameters for FVIII were calculated for 6/9 subjects randomized; in three subjects baseline anti-porcine FVIII inhibitors led to a lack of measurable FVIII activity. Mean C(max) appeared higher for OBI-1 (OSCA: 176.00 U dL(-1), standard deviation ± 88.00; chromogenic: 151.00 ± 31.51 U dL(-1)) than Hyate:C (OSCA: 82.3 ± 19.22 U dL(-1); chromogenic: 52.67 ± 13.8 U dL(-1)). Mean AUC also appeared higher for OBI-1 (OSCA: 2082.87 ± 1323.43 U h(-1) dL(-1) ; chromogenic: 1817.28 ± 625.14 U h(-1) dL(-1)) than Hyate:C (OSCA: 1177.8 ± 469.49 U h(-1) dL(-1); chromogenic: 707.61 ± 420.05 U h(-1) dL(-1)). Two infusion-related events occurred: one with Hyate:C, one with placebo. Four of five subjects without anti-porcine FVIII inhibitors at baseline remained porcine FVIII inhibitor negative 29 days after infusion. A single dose of OBI-1 appears to have higher bioavailability than Hyate:C in subjects with haemophilia A without measurable anti-porcine FVIII inhibitors, and is well tolerated. These results should be confirmed in a larger phase 2/3 study.

A B S T R A C T 2-Methylthio-ADP and its radioactive analogue [,_-32P]2-methylthio-ADP were synthesized and used to investigate platelet receptors for ADP. 2-Methylthio-ADP induced platelet aggregation and shape change, and inhibited cyclic AMP accumulation in platelets exposed to prostaglandin El. Compared with ADP, 2-methylthio-ADP was 3-5 times as active as an aggregating agent and 150-200 times as active as an inhibitor of cyclic AMP accumulation. Binding of [#-32P]2-methylthio-ADP to platelets was measured after centrifuging them through silicone oil to separate platelets from their suspension medium. Binding was reversible, saturable, and specific, with between 400 and 1,200 sites/cell in different platelet preparations. There was no evidence for a second class of binding sites with different affinity. The second order association rate constant was -3.5 X 10' M`so', and the first order dissociation rate was 0.024 s-', both measured at 230C. The dissociation equilibrium constant (-15 nM) was about three times higher than the concentration giving half-maximal inhibition of prostaglandin El-stimulated cyclic AMP accumulation in platelet-rich plasma. Binding was inhibited by ADP (Kj = 3.5 MM), ATP (7 AM), 2-azido-ADP (0.12 AM), inosine diphosphate (IDP, 150 AM), guanosine diphosphate (GDP, 350 MM), and AMP (800 MM). Binding of 2-methylthio-ADP was also blocked by the noncell-penetrating thiol reagent, p-mercuribenzene sulphonate, a reagent that blocks the inhibition of adenylate cyclase by ADP, but which does not block the ability of ADP to induce aggregation or platelet shape change. The amount of 2-methylthio-ADP bound at saturation was independent of pH in the range 6-8, but the affinity was reduced at pH 6 compared with pH 6.5-8.0. The dissociation constant was not temperature dependent in the range 320-40'C, whereas the rate of dissociation of 2-methylthio-ADP from platelets after the addition of an excess of ADP approximately doubled over this range. The activation energy for dissociation was -15 kcal/mol. Our results support the conclusion that platelets have a receptor for ADP, which inhibits cyclic AMP accumulation, and which has a sulphydryl group in the binding pocket. INTRODUCTION ADP has two distinguishable effects on blood platelets (1): firstly, it induces aggregation of the cell by stimulating a change in their shape and exposure of fibrinogen-binding sites, and, secondly, it inhibits the adenylate cyclase of membrane preparations of platelets and prevents the accumulation of cyclic AMP by intact platelets (2). Inhibition of aggregation by pros-

Hepatitis C virus (HCV) requires reverse transcriptase-polymerase chain reaction (RT-PCR) or branched DNA signal amplification assays to be detected in patient samples. Although conventional methods of RNA isolation are employed for samples of serum, plasma, and peripheral blood mononuclear cells (PBMCs), whole blood is generally considered an unsuitable source of RNA because of abundant RNases and polymerase inhibitors. Using a cationic surfactant, Catrimox-14, we adapted a procedure for RNA isolation from whole blood, plasma, and PBMCs that yields RNA template suitable for HCV RT-PCR. RNA isolation required less than 2 hr, and HCV sequences were easily detected in sample volumes of 50 microliters whole blood or plasma, and in less than 1 x 10(4) PBMC. Following the addition of blood to Catrimox, HCV RNA was stable in the mixture when incubated for at least 7 days at room temperature prior to RNA extraction. Comparison of whole blood HCV RNA and plasma HCV RNA from individuals with chronic hepatitis suggests that HDV RNA can be more reliably detected in whole blood. Three of 15 HCV antibody positive patients (20%) had HCV RNA present in whole blood but simultaneously obtained plasma samples were negative. Two of the HCV antibody negative individuals with chronic hepatitis contained HCV RNA in whole blood, yet one of these patient's plasma was negative for viral RNA. The Catrimox-14 method of RNA purification is useful for detecting HCV RNA in whole blood and blood subfractions, and provides a practical method of measuring plasma and PBMC HCV RNA from clinical specimens.

After surgery, enoxaparin, 40 mg once daily or 30 mg every 12 hours, is more effective than a regimen of 10 mg once daily to prevent deep venous thrombosis in patients having elective hip replacement surgery. The regimens of 40 mg once daily and 30 mg every 12 hours provided prophylaxis similar to the most effective drug treatments previously reported. The incidence of hemorrhagic episodes with the regimens of 40 mg once daily and 30 mg twice daily was higher than that observed with 10 mg once daily; however, major hemorrhage occurred in only 4% to 5% of patients receiving the higher-dose regimens. The risk-to-benefit ratio supports the use of enoxaparin as a therapeutic agent to prevent deep venous thrombosis in these patients.

Introduction: Development of inhibitors to human FVIII (hFVIII) significantly complicates the control of bleeding events in patients with haemophilia A. Aim: This prospective, multicentre, open-label, non-comparative, Phase II study evaluated the haemostatic activity of a recombinant B-domain-deleted porcine FVIII (r-pFVIII), in the treatment of non-life/non-limb-threatening bleeding in individuals with haemophilia A and FVIII inhibitors. Methods: Acute bleeding episodes in patients with pFVIII inhibitor titres <0.8 BU mL À1 were treated with 50 U kg À1 body weight r-pFVIII. Those with pFVIII inhibitor titres of >0.8 BU mL À1 received an initial calculated r-pFVIII loading dose followed by 50 U kg À1 treatment dose. Treatment continued at 6-hourly intervals until bleeding was determined, controlled or till a maximum of eight doses was reached. Results: All 25 bleeding episodes in nine patients (mean age: 23.7 years; range: 14-34 years) were controlled successfully with eight or fewer injections of r-pFVIII. The median time from bleeding onset to the administration of r-pFVIII was 5.7 h (range: 1.5-20.0 h). Twenty of the bleeding episodes (80%) were controlled with one treatment dose of r-pFVIII (with or without a loading dose, median dose: 200.8 U kg À1; range: 50-576 U kg À1) regardless of pFVIII level. r-pFVIII was well tolerated and no treatment-emergent serious adverse events were considered by the investigator to be related to r-pFVIII administration. Conclusion: The results suggest that FVIII replacement therapy with r-pFVIII could be a viable alternative to bypassing agents for the treatment of bleeding episodes in individuals with haemophilia A and FVIII inhibitors.

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